ADAM12 Inhibitors and Their Use Against Inflammation-Induced Fibrosis

ABSTRACT

The present invention relates to the field of fibrosis and inflammation and more particularly to the use of ADAM12 (A Disintegrin and Metalloproteinase 12) inhibitors to prevent or treat inflammation-induced fibrosis. The present invention also relates to the use of ADAM12 as a marker for inflammation-induced fibrosis and to the ablation of ADAM12 expressing cells as therapeutic approach to interfere with the development of pro-fibrotic cells.

FIELD OF THE INVENTION

The present invention relates to the field of fibrosis and inflammationand inflammation-induced fibrosis and more particularly to the use ofADAM12 (A Disintegrin and Metalloproteinase 12) inhibitors to prevent ortreat inflammation-induced fibrosis. The present invention also relatesto the use of ADAM12 as a marker for the inflammation-induced fibrosis.The present invention further relates to the ablation ofADAM12-expressing cells as a therapeutic approach for interfering withthe development of pro-fibrotic cells.

BACKGROUND OF THE INVENTION

Tissue injury initiates a scarring process involving stromal cellactivation and expansion. Initially beneficial, persistent stromal cellactivation and proliferation generate pro-fibrotic cells andaccumulation of extracellular matrix, which compromises organ recovery.Inflammation is an important initiator of the fibrosis but the cellularorigin of pro-fibrotic stromal cells and their mechanism of activationremain unclear.

During inflammation, stromal cells regulate lymphocyte recruitment andsurvival through the secretion of specific chemokines and cytokines,resulting eventually in the clearance of inflammation, this phase isusually named the acute phase of inflammation. Disruption of thishomeostasis can lead to chronic inflammation and its corollary of severeillnesses, such as tissue fibrosis.

Fibrosis is the formation of excess fibrous connective tissue during thereparative and reactive process following tissue damage andinflammation. As major components of fibrosis, pro-fibrotic stromalcells play a critical role in the maintenance of chronic inflammationand often preclude full organ recovery, and in some cases can induceorgan failure. This can lead to severe illnesses of major impact onpublic health such as liver cirrhosis, scleroderma, heart and pulmonaryfibrosis, atherosclerosis, and asthma. The formation of tissue fibrosisis currently considered to be an irreversible process, which is poorlymodulated by anti-inflammatory and immunosuppressive drugs. Themechanisms leading to the formation of tissue fibrosis has remainedelusive and therefore has prevented the elaboration of an adequatetherapeutic treatment.

The inventors have described previously the emergence of a stromalpopulation of cells (gp38+ lymphoid stromal cells) that massivelyproliferate and provide key survival, chemotactic and morphogenicfactors for the development of lymphocyte permissive tissue duringontogeny and inflammation (Peduto et al., Journal of Immunology, 2009,182:5789-5799). They have shown that these stromal cells are notrecruited from circulating precursors but develop from tissue-residentcells that remained to be characterized.

ADAMs (A Disintegrin And Metalloproteinase) are a family of cell surfacemultidomain proteins having key roles in the ectodomain shedding andprocessing of growth factors, cytokines, receptors, adhesion moleculesand other molecules from the plasma membrane. This process has emergedas an important posttranslational regulator of the function of manycleaved substrate proteins, including EGF-receptor ligands and TNFα. Inaddition to protease activity, several ADAMs possess cell binding andcell signalling properties due to their non-catalytic domains. More than30 members have been identified in the ADAM family with a broad tissuedistribution and have been involved in several cellular processes. Dueto their ability to rapidly affect key signalling activities betweencells and their environment, ADAM family members could conceivablycontribute to pathogenesis including tumorigenesis, especially if theirfunction is dysregulated. Therefore, they are making up the majority ofpharmaceutical targets currently undergoing preclinical and clinicalevaluation (Moss et al., “ADAMs: Targets for Drug Discovery”, CurrentPharmaceutical Design, 2009, 15(20):2270-1).

ADAM12 is an active protease involved in the Epidermal Growth FactorReceptor (EGFR) and insulin-like growth factor (IGF) receptorsignalling, and plays a role in tumor progression (Peduto et al.,Oncogene, 2006, 25:5462-5466). ADAM12 is also involved in severaldiseases such as arthrosis, cardiac hypertrophy and neurodegenerativediseases (Jacobsen & Wewer, Current Pharmaceutical Design, 2009, 15:2300-2310).

There is still a need to provide tools, markers and inhibitors forinflammation induced fibrosis.

SUMMARY OF THE INVENTION

The present invention provides new tools for the prevention, treatmentand diagnosis of fibrosis. In one aspect, the present invention concernsthe use of an ADAM12 inhibitor for preventing and/or treatinginflammation-induced fibrosis.

Yet another object of the invention concerns a composition forpreventing or treating inflammation-induced fibrosis comprising anADAM12 inhibitor and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method for preventing or treatinginflammation-induced fibrosis in a subject, comprising the step ofadministering the composition as defined above to the subject.

In another aspect, the present invention provides the use of ADAM12 asan early marker for inflammation-induced fibrosis.

In another aspect, the present invention provides the use of ADAM12 as amarker for activated stromal cells involved in inflammation-inducedfibrosis.

In another aspect, the present invention provides a method fordiagnosing inflammation-induced fibrosis in a subject, comprising:

-   -   detecting the expression of ADAM12 gene in stromal cells from a        biological sample of said subject, and    -   relating the expression of ADAM12 gene to the presence or the        advent of inflammation-induced fibrosis.

In another aspect, the present invention provides a method fordiagnosing inflammation-induced fibrosis in a subject, comprising:

-   -   determining the level of ADAM12 gene expression in stromal cells        from a biological sample of said subject, and    -   comparing this level with a reference level, whereby an        increased level of ADAM12 gene expression in said stromal cells        with respect to the reference level indicating the presence or        the advent of inflammation-induced fibrosis.

The present invention also concerns a method for detecting stromal cellsactivated upon tissue injury and inflammation and that will generatepro-fibrotic cells by detecting ADAM12 expression in the stromal cells,and relating the expression of ADAM12 gene to the presence of theseactivated stromal cells.

The present invention also concerns a bacterial strain comprising agenerated bacterial artificial chromosome (BAC) expressing a Crerecombinase and a fluorescent reporter EGFP under control of the Adam12gene, deposited on Aug. 28, 2009 under No I-4225 at the CNCM.

In another aspect, the invention provides a transgenic mouse comprisingthe BAC contained in the bacterial strain of the invention.

Still another object of the invention is to provide a screening methodfor identifying ADAM12 inhibitors, comprising the steps of:

-   -   inducing an inflammation response at a desired site of the        transgenic mouse of the invention,    -   applying at the site of inflammation a candidate compound to be        tested,    -   evaluating expression of the fluorescent reporter EGFP        polypeptide; and    -   identifying that the expression level of said fluorescent        reporter EGFP is inhibited, therefore indicating that the        candidate compound has the capacity of inhibiting the ADAM12        gene expression.    -   Another object of the invention concerns a cytotoxic compound        able to specifically kill ADAM12 expressing stromal cells for        use to prevent or treat fibrosis, wherein said compound        comprises a targeting molecule selected among an antibody or a        small interfering RNA specific for ADAM12, and a cytotoxic        molecule which is a toxin.

Yet another object of the invention concerns a composition forpreventing or treating inflammation-induced fibrosis comprising acytotoxic compound targeting ADAM12 expressing cells to kill them and apharmaceutically acceptable carrier. Such compound includes specificantibodies against ADAM12 coupled to a toxin.

The invention concerns also the use of the described transgenic mouse asa tool to detect activated stromal cells, as defined by the expressionof ADAM12, during acute or chronic inflammation, and more generally infibrosis-related diseases.

In another aspect, the present invention provides the use of ADAM12 as atarget to specifically kill stromal cells activated by inflammation andgenerating pro-fibrotic cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Generation of the BAC-transgenic mice ADAM12-cre-ires-egfp (A)and cell fate mapping strategy (B).

FIG. 2. ADAM12 expression (M12-CIG) and cell fate mapping ofADAM12-expressing cells (M12cre/RFP) in CFA-induced skin inflammation(A) and cardiotoxin-induced muscle inflammation (B) at 4 days for ADAM12expression and 15 days for fate mapping experiment. Co-staining withalpha-smooth muscle actin antibodies after 30 days (C). In (D),schematic description of ADAM12 expression (first peak) and fate mappingof ADAM12+ cells (second peak).

M: muscle; F: fibrosis; CFA: complete Freud's adjuvant; CX: cardiotoxin;GFP: green fluorescent protein; RFP: red fluorescent protein; α-SMA:alpha Smooth Muscle Actin; dapi labels nuclei.

FIG. 3. Gene expression analysis by qRT-PCR of FACS-sorted GFP+ cellsgenerated during inflammation. IL1beta, IL6, TNF alpha, TGF beta, CxCl1, CxCl 10 in (A), and Ctgf, Col1A, IGF 1, FGF 1 in (B).

FIG. 4. In vivo knock down of ADAM12 expression by RNA interferenceduring muscle injury and inflammation. Immunofluorescence analysis (A),Gene expression, as measured by quantitative RT-PCR (B); (upper panel:ear skin 6 days after CFA injection; lower panel: muscle 22 days afterCX injection), and fibrotic index (C) of inflamed tissues treated withADAM12 siRNAs at different times as indicated in results. In A, arepresentative picture of three independent experiments is shown. Thefibrotic index was calculated as the % of the injured area occupied bycollagen deposit. Error bars, s.d.; *P<0.05, unpaired t-test.

FIG. 5. DNA sequence (5′->3′ coding strand) of a polynucleotide whichencodes for CRE-IRES-EGFP-flag polypeptide. The first 1032 bp code forCRE, the following 581 bp code for IRES, the following 720 bp code forEGFP and the last 24 bp code for flag peptide.

FIG. 6. Strategy for inducible cell fate mapping (A). Sections ofinjured muscles 3 weeks after CX injection and DTA treatment inM12tTA/LC1/YFP mice were stained as indicated. The number of YFP⁺ cellsand CD45⁺ cells per field was counted in at least 10 sections of 3independent experiments (B). Fibrotic index was calculated as the % ofthe injured area occupied by collagen deposits (C). Error bars, s.d.;*P<0.05, **P<0.005, unpaired t-test.

FIG. 7. Sciatic nerve injury was performed in M12-CIG mice (A) andM12tTA/LC1/YFP mice (B). Sections of innervated muscle were stained asindicated in Material & Methods.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

“ADAM12” refers to “A Disintegrin And Metalloproteinase 12”, also knownas meltrin-alpha. There are 2 splice variants of human ADAM12, atransmembrane type (ADAM12-L) and a secreted type (ADAM12-S) (Mino etal, J. Surgical Oncology, 2009, 100: 267-272). In addition to proteaseactivity, ADAM12 possesses cell binding and cell signalling properties.The catalytic domain mediates processing of growth factors and cytokinesand has been involved in epidermal growth factor (EGF) and insulin-likegrowth factor receptor signalling. The disintegrin, cysteine-rich, andEGF-like domains mediate contacts with the extracellular matrix andother cells through interactions with integrins and syndecans. Thecytoplasmic domain interacts with intracellular signalling molecules(Jacobsen and Wewer, 2009). Adam12 or Adam12 refers to the gene encodingthe ADAM12 protein. In the context of present invention the term“ADAM12” will be used also for referring to the gene. The sequences ofthe gene and protein are in the prior art, the sequence of the murinegene is available for example from the BAC clone RP23-404F23 fromInvitrogen.“Stromal cells” refer to connective tissue cells of an organ. These arethe cells that make up the support structure of biological tissues andsupport the parenchymal cells. Fibroblasts, immune cells, pericytes,endothelial cells and inflammatory cells are the most common types ofstromal cells. In this invention, the term “stromal cells” will be usedto refer to non-hematopoietic stromal cells only.“Activated stromal cells” refer to stromal cells that are activated andproliferate during inflammation and fibrosis.“Pro-fibrotic stromal cells” refer to subsets of activated stromal cellsthat promote the formation of fibrosis.“Diseases related to fibrosis” refer to diseases where fibrosis is animportant component of the pathogenesis, such as acute or chronicinflammation, and neuromuscular or neurodegenerative diseases.“Inflammation-induced fibrosis” relates to fibrosis developing duringinflammatory diseases i.e. diseases related to acute or chronicinflammation (caused by tissue injury, pathogen infections or toxicagents) or as a consequence. Fibrosis is the formation of excess fibrousconnective tissue during the reparative and reactive process ofinflammation. In the context of the present invention, the term“fibrosis” will also be used with the same meaning as“inflammation-induced fibrosis”.“Preventing inflammation-induced fibrosis” relates to a process by whichthe symptoms of fibrosis are obstructed or delayed.“Treating inflammation-induced fibrosis” relates to a process by whichthe symptoms of fibrosis are alleviated or eliminated.“A pharmaceutically acceptable carrier” relates to a vehicle forcontaining the ADAM12 inhibitor of the invention that can beadministered to a subject without adverse effects. Suitablepharmaceutically acceptable carriers known in the art include, but arenot limited to, sterile water, saline, glucose, dextrose or bufferedsolutions. Carriers may include auxiliary agents including, but notlimited to, diluents, stabilizers (La, sugars and amino acids),preservatives, wetting agents, emulsifying agents, pH buffering agents,viscosity enhancing additives, colors and the like.“A subject” refers to an animal, for instance a mammal, (e.g. a human).“EGFP” refers to Enhanced Green Fluorescent Protein.“CX” refers to cardiotoxin.“CFA” refers to complete Freud's adjuvant.

2. ADAM12 Inhibitors and Uses Thereof

The present inventors have unexpectedly found that it is advantageous toinhibit ADAM12 in the prevention or treatment of fibrosis.

Accordingly, it is an object of the invention to use compounds or agentsthat specifically inhibit ADAM12. As used herein, the term “inhibit”when referring to ADAM12 refers to the inhibition of the Adam12 geneexpression or to the activity of the ADAM12 protein. In other words, itis meant that the inhibitor contemplated by the present invention hasthe ability to interfere with the function of the ADAM12 gene or itsrelated proteinic product in such a way as to decrease expression of theADAM12 gene or to reduce the level or activity of the product of theADAM12 gene. With respect to the contemplated inhibitors used inaccordance with the present invention, the expression “specificallyinhibits” refers to a compound or agent that inhibits the expression ofthe ADAM12 gene or to reduce the level or activity of the product of theADAM12 gene, but which does not substantially inhibit the expression ofanother gene or reduce the level or activity of the product of othergenes.

Inhibitors which inhibit the activity of a ADAM12 gene include but arenot limited to compounds or agents that inhibit transcription of thegene, compounds or agents that inhibit processing of the transcript ofthe ADAM12 gene, compounds or agents that reduce the stability of thetranscript of the ADAM12 gene, and compounds or agents that inhibittranslation of the mRNA transcribed from the ADAM12 gene.

Inhibitors which inhibit the biological activity of ADAM12 proteininclude but are not limited to compounds or agents that inhibit itsproteolytic activity, its adhesion activity and/or its cytoplasmicsignal transduction activity.

As one skilled in the art will appreciate, inhibitors contemplated bythe present invention may be, but are not limited to, an antisenseoligonucleotide, a siRNA, a miRNA, a small organic molecule, an enzyme,an antibody, a peptide or a polynucleotide encoding a polypeptide.

For example, small interfering RNA specific for ADAM12 which arecommercialized by Invitrogen may be useful within the scope of thepresent invention, such as Stealth Select RNAi siRNA specific for humanADAM12 (catalog references: 1299003 and 1299001). Small interfering RNAsalso include ADAM12 siRNA (h), called sc-41414 provided by Santa CruzBiotechnology, inc. which is a target-specific 20-25 nt siRNA designedto knock down human gene expression.

By “antibodies” it is meant herein any type of antibody and inparticular polyclonal antibodies, monoclonal antibodies, orantibody-like molecules, all which are specific to ADAM12.

The term “monoclonal antibody” encompasses:

-   -   monospecific antibodies i.e., molecules wherein the two antigen        binding sites (domains formed by the VH regions or by the        interaction of the VH and VL regions, and interacting with the        immunogen) recognize and bind the same immunogen.    -   trifunctional antibodies i.e., bispecific molecules as disclosed        hereinafter and further having an Fc region (CH2 and CH3        domains) of any origin, particularly of human origin.

The term “antibody-like molecule” refers to a molecule having all orpart of the variable heavy and light domains of an antibody, but devoidof the conventional structure of a four-chain antibody, and conservingnevertheless the capacity to interact with and bind an immunogen. In aparticular embodiment of the invention, an antibody-like molecule is afragment of an antibody and in particular comprises the CDR1, CDR2 andCDR3 regions of the VL and/or VH domains of a full length antibody.

The term “antibody-like molecule” encompasses in particular:

-   -   scFv, i.e., a VH domain genetically associated (optionally via a        linker) to a VL domain, as well as molecules comprising at least        one scFv, such as Bis ScFv molecules (two ScFv having same or        different antigen binding site(s) linked together (optionally        via a linker));    -   diabody molecules i.e., the heavy chain variable domain derived        from a first antibody (a first VH domain (VH1)) connected to the        light chain variable domain derived from a second antibody (VL2)        on the same polypeptide chain (VH1-VL2) connected by a peptide        linker that is too short to allow pairing between the two        domains on the same chain, interacting with the heavy chain        variable domain of derived from a second antibody (VH2)        connected to the light chain variable domain derived from a        first antibody (a first VL domain (VL1)) on the same polypeptide        chain (VH2-VL1), wherein VL1 and VH1 form a first        antigen-binding site and VL2 and VH2 form a second antigen        binding site (recognizing and/or binding a similar or a        different immunogen from the first binding antigen binding        site);    -   bispecific molecules i.e., molecules in which the two antigen        binding sites of a Fab₂ fragment (variable and CH1 domains of        light and heavy chains) interact with different immunogens).    -   trispecific molecules i.e., molecules in which the two antigen        binding sites of a Fab₃ fragment (variable and CH1 domains of        light and heavy chains) interact with different immunogens);    -   VHH (VH domain of functional antibodies naturally devoid of        light chains) i.e., a VH domain which has the capacity to        interact as such with an immunogen, without the presence of a        variable light domain (VL).    -   functional fragments of an antibody or an antibody-like molecule        as defined herein, provided that these fragments retain the        ability to specifically interact with subset(s) of cells and/or        cell surface molecule(s). These fragments include Fv fragments        (non-covalent association of the VH and VL domains of the        invention) and Fab fragments.

Polyclonal Antibodies are for instance, antibodies provided by SantaCruz Biotechnology, include ADAM12 (S-18) called sc-16526 or ADAM12(C-20) called sc-16527, which are affinity purified goat polyclonalantibodies raised against a peptide mapping within an internal region ofADAM12 of human origin or ADAM12 (H-210) called sc-25579, a rabbitpolyclonal antibody raised against amino acids 700-909 of ADAM12 ofhuman origin.

Monoclonal antibodies are for example 6E6, 8F8 and 6C10 as described inKronqvist et al., American Journal of Pathology 161, 5, 2002 and furtherdescribed in Gilpin et al., J. Biol. Chem., 2002, 160: 1895-1903; Iba etal., Am. J. Pathol., 1999, 154: 1489-1501; Iba et al., J. Cell Biol.,2000, 149:1143-1156.

The inhibitory compounds may be those that are known to affect ADAM12proteolytic activity including but not limited to: hydroxamateinhibitors, thiol amides which exhibit collagenase inhibition activity(U.S. Pat. No. 4,595,700), N-carboxyalkyl derivatives containing abiphenylethylglycine which inhibit MMP-3, MMP-2 and collagenase (WO95/29689 by Durette, et al.), lactam derivatives which inhibit matrixmetalloproteases, TNF-alpha and aggrecanase (U.S. Pat. No. 6,495,699),tricyclic sulfonamide compounds (U.S. Pat. No. 6,492,422), the compoundONO-4817 from Ono Pharmaceutical Co. Ltd., Osaka, Japan (Mori et al.,2002, Anticancer Res., 22(6C):3985-8) and the collagenase inhibitorsGM6001 (trade name Galardin™) and GM1489 (a derivative of GM6001) (U.S.Pat. No. 6,759,432). Specific examples of hydroxamic acid-basedmetalloprotease inhibitors include the compounds “5A”[NHOHCOCH₂CH(i-Bu)CO-tryptophan-NHMe], “21A”[NHOHCOCH₂CH(i-Bu)CO-tryptophan-NHCHMePh], “39A”[HOOCCH2CH(i-Bu)CO-tryptophan-NHCHMePh], “51209”[NHOHCOCH2CH(i-Bu)CO-tyrosine-OMeNHMe], UL001 [HSCH₂CH(CH₂CH(CH₃)₂)CO-Phe-Ala-NH₂] and MP506 (Elastin Products Company,Inc.) (U.S. Pat. Nos. 5,773,438 and 5,892,112). Other compounds, such asKB—R7785, could act as inhibitors of ADAM12 (Akasura M. et al., Nat.Med., 2002, 8, 35.). The matrix metalloprotease inhibitor SB-3CT is alsoexpected to inhibit ADAM12. Additional metalloprotease inhibitorsexpected to be useful as ADAM12 inhibitory compounds include the variouscompounds disclosed in U.S. Pat. Nos. 6,500,847; 6,268,379; 5,968,795;5,892,112; 5,872,152; 4,681,894; 4,943,587 and WO 06/014903. Fourselective ADAM12 inhibitors have been described by Oh et al., 2004,Bioorg Med Chem. Lett., 14(24):6071-6074, and called the compounds 5,11, 14, and 16, having the following structures

Another aspect of the present invention relates to compositions fortreating or preventing fibrosis in a subject. The composition of thepresent invention advantageously comprises at least one inhibitorymolecule as defined above. The composition of the invention furthercomprises a pharmaceutically acceptable carrier.

In a related aspect, the invention provides a method for treating orpreventing fibrosis in a subject. The method comprises the step ofadministering to said subject in need thereof a composition of theinvention.

The amount of the inhibitors of the composition of the invention ispreferably a therapeutically effective amount. A therapeuticallyeffective amount of the contemplated component is the amount necessaryto allow the same to perform its inhibitory role without causing overlynegative effects in the subject to which the composition isadministered. The exact amount of the inhibitors to be used and thecomposition to be administered will vary according to factors such asthe form of fibrosis being treated, the type and age of the subject tobe treated, the mode of administration, as well as the other ingredientsin the composition.

The composition of the invention may be given to the subject throughvarious routes of administration. For instance, the composition may beadministered in the form of sterile injectable preparations, such assterile injectable aqueous or oleaginous suspensions. These suspensionsmay be formulated according to techniques known in the art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparations may also be sterile injectable solutions orsuspensions in non-toxic parenterally-acceptable diluents or solvents.They may be given parenterally, for example intravenously,intramuscularly or sub-cutaneously, by injection, by infusion or per os.Suitable dosages will vary, depending upon factors such as the amount ofeach of the components in the composition, the desired effect (short orlong term), the route of administration, the age and the weight of thesubject to be treated. Any other methods well-known in the art may beused for administering the composition of the invention.

As one skilled in the art will understand, the administration of ADAM12inhibitors during acute and/or chronic inflammation, includinginfectious or inflammatory diseases is particularly useful to limit thegeneration of fibrosis and scar tissue. As fibrosis correlates with poororgan recovery, the inhibition of ADAM12 is expected to increase thefunctionality of the inflamed organ (such as muscle regeneration in thecase of muscle inflammation). This will also decrease leukocyteinfiltration, aiming at restoring tissue homeostasis and resolvinginflammation. Furthermore, since stromal cells represent the reservoirfor several infectious agents, such as Chikungunya virus in the muscle,Hepatitis virus in the liver, and Influenza A virus in the lung, theadministration of agents that inhibit ADAM12 might be particularlyuseful to modulate the course of infection of viruses targeting stromalcells, by limiting the expansion of the stromal compartment.

According to the invention, the ADAM12 inhibitor as defined herein istherefore for use in prevention and/or treatment of fibrosis-relateddiseases as liver cirrhosis, scleroderma, heart and pulmonary fibrosis,atherosclerosis and asthma.

3. Screening Method for Inhibitors of ADAM12 Gene Expression

The invention provides a polynucleotide which consists of a generatedbacterial artificial chromosome (BAC) expressing a Cre recombinase and afluorescent reporter protein: EGFP (Enhanced Green Fluorescent protein)under control of the murine Adam12 gene, and a bacterial straincomprising such polypeptide deposited on Aug. 28, 2009 under No I-4225at the CNCM.

In another aspect, the invention provides a transgenic mouse whichcomprises the polynucleotide described above and contained in thebacterial strain of the invention.

Still another object of the invention is to provide a screening methodfor identifying ADAM12 inhibitors, comprising the steps of:

-   -   inducing an inflammation response at a desired site of the        transgenic mouse of the invention,    -   applying at the site of inflammation a candidate compound to be        tested,    -   evaluating expression of the fluorescent reporter EGFP        polypeptide; and    -   identifying that the expression level of said fluorescent        reporter EGFP is inhibited, whereby indicating that the selected        compound has the capacity of inhibiting the Adam12 gene        expression.

The screening method may further comprise the step of assessing otherparameters of inflammation response with and without the compound, asleukocytes (CD45+ cells) infiltration and pro-inflammatory cytokinessuch as IL1 beta, IL6, TGF beta or TNF alpha.

As one skilled in the art will understand, the candidate compounds willadvantageously be tested to confirm that they do not inhibit theexpression level of said fluorescent reporter EGFP per se. Therefore, itwill be understood that if the tested compound does not inhibit theexpression level of said fluorescent reporter EGFP but such expressionis yet inhibited according to the screening method of the presentinvention, one will understand that the selected compound does have thecapacity of inhibiting the ADAM12 gene expression.

The screening method of the invention thus allows identification ofcompounds which interfere directly with the activity of the ADAM12promoter or which interfere with components of the signal transductionpathway of stromal cells during inflammation upstream from ADAM12.

The screening method of the invention can also be performed with atransgenic mouse which is the heterozygous progeny from crossingADAM12-CIG mouse with the knock-in Rosa26 mouse (Luche et al., “Faithfulactivation of an extra-bright red fluorescent protein in “knock-in”Cre-reporter mouse ideally suited for lineage tracing studies”. Eur J.Immunol. 2007 January; 37(1):43-53). With such a transgenic mouse, thescreening method may further comprise a step of evaluating expression ofthe fluorescent reporter RFP polypeptide. As for EGFP, if the expressionlevel of RFP is decreased with the compound when compared to the controlexpression level of RFP, one can conclude that the tested compound hasthe capacity of inhibiting the ADAM12 gene expression, therefore alsoits proteinic activity.

4. ADAM12 and Use as a Marker for Fibrosis

After transient expression during embryogenesis, ADAM12 is notexpressed, or expressed at low levels, in most normal adult tissuesunder homeostatic conditions. The invention relies on the finding by theinventors that in mouse models for inflammation by skin, muscle or nerveinjury, pro-fibrotic stromal cells develop from a pool of progenitorcells re-expressing ADAM12, which are activated at the onset ofinflammation and massively proliferate during the first days ofinflammation, post injury. These cells produce a large amount ofpro-inflammatory cytokines and growth factors and can be detected up to4 weeks after induction of inflammation. By genetic fate mapping, theinventors observe that inflammation-induced fibrosis is derived fromstromal cells re-expressing ADAM12 following tissue injury andinflammation.

Therefore, the present invention further provides the use of ADAM12 asan early marker for inflammation-induced fibrosis.

In a related aspect, there is provided a method for diagnosing fibrosisin a subject. Such a method comprises the step of evaluating if theAdam12 gene is expressed in stromal cells, either by detecting the mRNAlevel or the protein level. This diagnosis method will indicate that thetested subject suffers or will suffer from fibrosis.

In a related aspect, there is provided a method for diagnosinginflammation-induced fibrosis in a subject comprising:

-   -   detecting the expression of Adam12 gene in stromal cells from a        biological sample of said subject,    -   relating the expression of Adam12 gene to the presence or the        advent of inflammation-induced fibrosis.

Alternatively, is provided a method for diagnosing inflammation-inducedfibrosis in a subject, comprising:

-   -   determining the level of Adam12 gene expression in stromal cells        from a biological sample of said subject,    -   comparing this level with a reference level, whereby an        increased level of Adam12 gene expression in said stromal cells        with respect to the reference level indicating the presence or        the advent of inflammation-induced fibrosis.

According to another embodiment, the present invention is directed to amethod for detecting the presence of stromal cells specificallyactivated upon tissue injury and inflammation and responsible forinflammation-induced fibrosis in a subject comprising:

-   -   detecting the expression of ADAM12 gene in stromal cells from a        biological sample of said subject; and    -   relating the expression of ADAM12 gene to the presence of        stromal cells activated upon tissue injury and inflammation and        responsible for inflammation-induced fibrosis.

All the above-mentioned diagnosing and detecting methods of theinvention may advantageously further comprise a step of isolatingstromal cells, before the step of detection.

According to the invention, the stromal cells which are to be analysedcan be from a biopsy. A biopsy according to the invention may be fromany organ or tissue. The biopsy is for example a biopsy of muscle, lung,skin, liver or nerve.

The methods of the invention may be carried out in vitro or ex vivo.Indeed and as one skilled in the art will appreciate, the step ofdetecting the expression of ADAM12 gene may be carried out in vitro orex vivo.

By “expression of ADAM12 (or Adam12) gene, it is meant an expressionwhich is above the expression of ADAM12 in normal stromal cells underhomeostatic conditions, used as a control. Preferably, the normalstromal cells used as control are from the same adult tissue as the oneof stromal cells of the biological sample to test. Moreover, accordingto these embodiments of the invention, the sole detection of the ADAM12gene over-expression in stromal cells is indicative of the presence ofactivated stromal cells that could lead to fibrosis. Indeed, asmentioned above, ADAM12 expression in normal stromal cells from adulttissues under homeostatic conditions is very low, ADAM12 mRNAs andproteins are thus not detectable, or detected at low levels, in normalstromal cells by using standard methods, (e.g. Western blot andimmunohistology as detection means for proteins, and Northern blot or insitu hybridization as detection means for RNA), or as sensible or lesssensible detection techniques.

In the methods of invention, the expression of ADAM12 gene is determinedeither by the detection of RNA transcripts from ADAM12 gene or bydetection of proteins produced from ADAM mRNAs.

Detection of RNA transcripts can be performed with standard techniquessuch as Northern blot, in situ hybridization or quantitative RT-PCR.

When the detection of the expression of ADAM12 gene is performed by RNAdetection, the “reference level”, used during the comparison step, isthe level of ADAM12 RNA in normal, healthy stromal cells of the subjectto be tested, or of another subject. More preferably, the normal healthystromal cells used as reference are from the same tissue as the one ofstromal cells of the biological sample to test.

Alternatively, when the level of proteins corresponding to ADAM12 RNAsis to be determined, advantageous techniques to be employed are Westernblots and in situ immunoassays. The “reference level”, used during thecomparison step, is the level of ADAM12 protein in normal, healthystromal cells of the subject to be tested, or of another subject. Morepreferably, the normal healthy stromal cells used as reference are fromthe same tissue as the one of stromal cells of the biological sample totest.

By an “increased level”, it is to be understood that the level issignificantly increased, i.e. that the difference between the measuredlevel and the reference level is significant from a statistical point ofview, that is the difference is greater that the standard deviation ofthe measured levels. The difference is thus advantageously greater thanthe error inherent in the measurement and greater than the variationsobserved between normal stromal cells. More advantageously, theincreased level is at least 3 times higher than the reference level.

The present invention further provides kits for use within the abovedescribed diagnostic and detecting methods. Such kits typically comprisetwo or more components necessary for performing a diagnostic assay.Components may be compounds, reagents, containers and/or equipment. Forexample, a kit may be designed to detect the level of mRNA encoding anADAM-12 protein in a biological sample. Such kit generally comprises atleast one oligonucleotide probe or primer that hybridizes to apolynucleotide encoding an ADAM-12 protein. Such an oligonucleotide maybe used, for example, within a PCR or hybridization assay. Additionalcomponents that may be present within such kits include a secondoligonucleotide and/or a diagnostic reagent or container to facilitatethe detection of a polynucleotide encoding an ADAM-12 protein. Such kitsmay also, or alternatively, contain a monoclonal antibody or fragmentthereof that specifically binds to an ADAM-12 protein. Such antibodiesor fragments may be provided attached to a support material, as known toone skilled in the art. One container within such a kit may also containa detection reagent that contains a reporter group suitable for director indirect detection of antibody binding. One or more additionalcontainers may enclose elements, such as reagents or buffers, to be usedin the assay.

5. Method for Killing ADAM12-Expressing Stromal Cells

In another aspect, the present invention provides the use of ADAM12 as atarget to specifically kill stromal cells activated duringinflammation-induced fibrosis.

This aspect of the invention relies on the finding by the inventors thatin mouse models for muscle injury and inflammation, the genetic ablationof ADAM12-expressing cells decreases inflammation and fibrosis inchronic lesions.

Therefore, the present invention provides a cytotoxic compound for useto kill ADAM12-expressing cells. Therefore the present inventionprovides a cytotoxic compound for use to prevent or treatinflammation-induced fibrosis.

A cytotoxic compound of the invention may comprise a targeting moleculeand a cytotoxic molecule and is able to specifically killADAM12-expressing stromal cells.

The targeting molecule specifically recognizes ADAM12-expressing cellsby binding to ADAM12 protein or ADAM12 mRNA present in or at the surfaceof the cell. The targeting molecule is, for example, an antibody raisedspecifically against ADAM12 protein or any part thereof, or a smallinterfering RNA specific for ADAM12. Examples of antibodies and smallinterfering RNA specific for ADAM12 are described above.

The cytotoxic molecule is, for example, a toxin, such as Pseudomonasexotoxin (Wolf et al., Int J Med Microbiol., 2009 March; 299(3):161-76.Epub 2008 Oct. 23), deglycosilated ricin A-chain (dgA) (Schnell et al.,Ann Oncol., 2003 May; 14(5):729-360) or the cytotoxic drug maytansinoid1 (DM1) (Kovtun et al., Cancer Res. 2010 Mar. 15; 70(6):2528-37. Epub2010 Mar. 2).

The targeting molecule and the cytotoxic molecule are associated in thecytotoxic compound by chemical coupling.

The invention is also directed to the cytotoxic compound as definedherein for use in the prevention and/or the treatment of fibrosis.

The invention also relates to a cytotoxic compound as defined herein,for use in combination with another anti-inflammatory drug to prevent ortreat fibrosis.

As ADAM12 is involved in other pathologies than fibrosis-relateddiseases, such as arthrosis, cardiac hypertrophy, neurodegenerativediseases, neuromuscular diseases and cancer, the cytotoxic compound asdefined herein can be used for preventing or treating these diseases.

The present invention will be more readily understood by referring tothe following examples. These examples are illustrative of the widerange of applicability of the present invention and are not intended tolimit its scope. Modifications and variations can be made thereinwithout departing from the spirit and scope of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice for testing of the present invention,the preferred methods and materials are described. Several experienceshave been repeated, with some changes. All references cited in thepresent application are incorporated by reference.

EXAMPLES

The present inventors now present a role for ADAM12 in the inflammationprocess such as inflammation resulting from tissue injury, and moreparticularly in inflammation-induced fibrosis and generation ofpro-fibrotic cells. It is shown that ADAM12 expression increases uponskin, muscle and nerve injury (tissue injury is a way to induceinflammation) as activated stromal cells start to expand, before beingdown regulated in later fibrotic lesions. These cells produce a largeamount of pro-inflammatory cytokines and/or growth factors. Genetic fatemapping indicate that most inflammation-induced fibrosis is progeny ofADAM12-expressing cells that massively proliferate in the first daysafter induction of inflammation caused by tissue injury. In vivotreatment with ADAM12 specific small interfering RNA in mice leads to areduction of inflammation-induced fibrosis. It is also shown thatspecific ablation of ADAM12-expressing cells generated post injury issufficient to mitigate the pro-fibrotic process. Accumulation ofalpha-SMA+ pro-fibrotic cells, and collagen are significantly reduced.In accordance with a role for stromal cells in the recruitment ofleucocytes, the inhibition of ADAM12 or the ablation of ADAM12+ cellslead locally to a decrease in the recruitment of CD45+ cells in theinflamed tissues. The inventors' results demonstrate that stromal cellsand the immune system communicate through a cellular crosstalk togenerate an effective immune response. Hence the overactivation ofstromal cells, through a pathway involving ADAM12, disrupts homeostasisby maintaining the recruitment of immune cells.

These results indicate that ADAM12 plays an essential role in thedevelopment of inflammation-induced fibrosis, and suggest that theoveractivation of stromal cells, through a pathway involving ADAM12,might be involved in maintaining the recruitment of immune cells.

Material & Methods Mice.

Generation of ADAM12-CIG mice (M12-CIG). The coding sequence forCRE-IRES-EGFP-flag (FIG. 5-SEQ ID NO:1), including the stop codon and apolyA sequence, was generated by overlap PCR and inserted into exon 1 ofAdam12 mouse gene in place of the endogenous ATG translation startcodon, on a 200-kb Bacterial Artificial Chromosome (BAC) (cloneRP23-404F23 from Invitrogen) carrying at least 70 kb, preferably 80 kbof sequence upstream of the Adam12 translation start site. Generation ofADAM12-tTA-ires-Dtr^(TG) mice (M12-tTA): the coding sequence fortTA-ires-Dtr, including a poly A sequence, was generated by overlap PCRand inserted into exon 1 of Adam12 in place of the endogenous ATGtranslation start codon, on the same 200 kb BAC as described above.

Growth Conditions and Viability of Bacteria BAC-M12-CIG

1. If starting from a frozen stock of bacteria, streak a few crystalsonto plates LB containing 12.5 μg/ml chloramphenicol

2. Incubate 0/N at 37° C.

3. Perform direct colony PCR on 4 colonies with primers specific for thegene cre, as described:

A. Pick colonies:

-   -   Pick a colony with a 200 μl tip on a pipette    -   Agitate it in 5 ml LB miniculture+12.5 μg/ml Chloramphenicol    -   Wash the tip in 100 μl water

B. Perform PCR with 5 μl of water in 25 μl reactions:

-   -   H2O to 25 μl    -   Buffer 10×2.5 μl    -   MgCl₂ 50 mM 0.75 μl    -   dNTPs 10 mM 0.5 μl    -   Regular Taq 0.4 μl    -   Primers 25 mM 0.5 μl each    -   Substrate (water+colony) 5 μl

PCR Program:

1.96° C. 5 min

2. 96° C. 30 sec

3. 58° C. 30 sec

4. 72° C. for 1 min

5. Repeat steps 2-4 34x

6. 72° C. 7 min

7. 4° C. for ever

Primers Sequence for Detection of cre:

119 sequence (5′-3′): (SEQ ID NO: 2) TAA AGA TAT CTC ACG TAC TGA CGG TG120 sequence (5′-3′): (SEQ ID NO: 3) TCT CTG ACC AGA GTC ATC CTT AGC

C. Load PCR reactions onto 1% normal agarose. Positive band 0.25 kb

4. Grow the positive minicultures 0/N in 3 ml LB+12.5 μg/mlchloramphenicol5. The next day, freeze aliquots of positive minicultures and/or put 1ml in 500 ml LB+Chloramphenicol (grow again O/N at 37° C. withagitation) for preparation of BAC DNA.

After modification of the ADAM12 BAC in vitro by homologousrecombination, M12-CIG and M12-tTA BACs containing the insertedsequences were purified and injected into fertilized eggs implanted intofoster mothers by homologous recombination as previously described(Sparwasser et al., “General method for the modification of differentBAC types and the rapid generation of BAC transgenic mice”. Genesis.2004 January; 38(1):39-50).

Cell fate mapping experiments were performed by crossing M12-CIG micewith the knock-in Rosa26+/Fl RFP mice (Luche et al., “Faithfulactivation of an extra-bright red fluorescent protein in “knock-in”Cre-reporter mice ideally suited for lineage tracing studies”. Eur JImmunol. 2007 January; 37(1):43-53). For inducible cell fate mapping,M12-tTA^(TG) mice were crossed with doxycycline-dependent LC1-Cre mice(Schonig, K., Schwenk, F., Rajewsky, K. & Bujard, H. Stringentdoxycycline dependent control of CRE recombinase in vivo. Nucleic AcidsRes. 30, e134., 2002) and then further crossed with Rosa26^(+/loxP-YFP)mice (Srinivas et al. Cre reporter strains produced by targetedinsertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4,2001).

All mice were kept in specific pathogen-free conditions and animalexperiments were approved by the committee on animal experimentation ofthe Institut Pasteur and by the French Ministry of Agriculture.

Mice Treatment.

Mice were anaesthetized with 100 μl of Xylazine/Ketamine. To induce earskin inflammation, ears were injected intradermally with 25 μl ofComplete Freud Adjuvant (CFA). To induce muscle inflammation, TibialisAnterior (TA) muscles were injected locally with 50 μl of 10 μMcardiotoxin (CX) in PBS. To perform nerve crush injury, the sciaticnerve in the thigh was exposed and slightly crushed with forceps.

At different time points after treatment, the inflamed ears (includingthe muscle base), the cardiotoxin-treated TA muscles and the injurednerve were removed and processed for histology.

M12tTA/LCI/YFP triple transgenic mice were treated with doxycycline(Sigma Aldrich) at 1 mg/ml in drinking water containing 5% Sucrose tostop fate-mapping labelling.

To delete ADAM12⁺ cells, M12tTA/LCI/YFP mice were injected with DTA(Diphtheria toxin subunit A) at days 4, 7 and 10 after CX injection, andtissues were collected after 3 weeks.

Cells Isolation and FACS.

To isolate GFP cells, inflamed muscles and ears from adult mice wereremoved and washed with PBS (Ca/Mg free), cut into 1 mm pieces andincubated at 37° C. for 30 min in a digestion medium composed of DMEM(Gibco) containing 1 mg/ml collagenase D (Roche), blendzyme III (Roche)and 1 U/ml DNase 1 (Invitrogen). After 30 minutes, tissues were washedwith DMEM 10% FCS and the supernatant was collected. Remainingundigested tissues were subjected to 1 or 2 additional cycles of 30 mindigestion, washed, mixed with the collected supernatants and pressedthrough a 100-μm mesh.

Knock Down of ADAM12 by RNAi.

To induce the knock down of ADAM12 gene expression, inflamed tissueswere treated with Stealth RNAi small interfering RNA (siRNAs) targetingmouse ADAM12 (purchased as MSS273021 from Invitrogen). Effective ADAM12knock down was first validated in vitro by screening three pre-designedADAM12 specific siRNAs in mammalian stromal cell lines (OP9). The siRNAinducing the best ADAM12 knock down in vitro was chosen for subsequentin vivo studies. In vivo knock down in tissues was performed followingmanufacturer's recommended procedure. Briefly, in vivo quality purifiedsiRNAs were mixed with Invivofectamine™ (Invitrogen) to obtain a finalconcentration of 20 mg/ml RNAi duplex. The lipid complex was thendiluted with 10 ml 5% glucose and concentrated using Amicon Ultra-15Centrifugal Device™ with Ultracel-50 Membrane™ (Millipore). Theretentate containing the Invivofectamine-RNAi complex was collected,resuspended in 5% glucose solution and sterilized by filtering through a0.22 μm filter prior to injection. siRNAs were delivered locally byinjecting 15 μl of 20 mg/ml siRNA solution in the ear skin at day 1 andday 3 after CFA injection; and in the tibialis anterior muscle at days2, 7, and 12, or at days 2, 7, 12, and 17 after cardiotoxin-inducedmuscle inflammation through Injection of cardiotoxin. CFA-inflamed earstreated with siRNAs were collected at day 6 and cardiotoxin-inflamedmuscles were collected after three weeks around day 22. Controlexperiments were performed by injecting scrambled (cont) siRNAs duplexwith similar GC content.

Gene Expression Analysis.

To obtain RNA for gene expression analysis by real time RT-PCR, 500-5000cells were directly FACS-sorted into vials containing RLT buffer(Qiagen) supplemented with 3-mercaptoethanol (see part “Cells isolationand FACS” for detailed protocol), and total RNA was extracted usingRNeasy Micro Kit™ (Qiagen). The quality of total RNA was assessed usingthe 2100 Bioanalyzer system (Agilent Technologies). 250-500 pg of highquality total RNA was subjected to one linear mRNA amplification cycleusing the MessageBooster Kit for qRT-PCR (Epicentre Biotechnologies).50-100 ng of amplified mRNA was transcribed into cDNA using SuperscriptIII reverse transcriptase (Invitrogen).

In RNAi knock down experiments of inflamed organs, 2 mm² of tissue weresnapped-freezed and total RNA was extracted with Rneasy Kit™ (Qiagen).The quality of total RNA was assessed using the 2100 Bioanalyzer™ system(Agilent Technologies). When necessary, 250-500 pg of high quality totalRNA was subjected to one linear mRNA amplification cycle using theMessageBooster Kit™ for qRT-PCR (Epicentre Biotechnologies). 50-100 ngof amplified mRNA were then converted to cDNA using Superscript III™(Invitrogen).

All procedures were performed according to the manufacturer's protocols.The expression of genes of interest was measured by quantitative realtime PCR which was performed using RT² qPCR Primer sets and RT²SYBR-Green master mix (SABiosciences) on a PTC-200 thermocycler equippedwith a Chromo4 detector (Bio-Rad Laboratories). Data was analyzed usingOpticon Monitor Software (Bio-Rad Laboratories). CT values werenormalized to the mean CT values obtained for the two house keepinggenes Hsp90 and Gapdh. All primers used in qRT-PCR were purchased fromSABiosciences.

Immunofluorescence Histology.

Tissue processing and immunofluorescence protocol have been describedpreviously (Peduto et al., 2009). Briefly, tissues were fixed 0/N at 4°C. in 4% paraformaldehyde (Sigma), then washed 0/N in PBS, incubated for2-4 hours in a solution of 30% sucrose (Sigma) until the samples sank,embedded in OCT compound 4583™ (Sakura Finetek), frozen in a bath ofisopentane cooled with liquid nitrogen and stocked at −80° C. Frozenblocks were cut at 8 μm thickness and sections were processed forstaining: after blocking with 10% bovine serum in PBS containing 1%Triton (PBS-XG) for 1 hour at room temperature, slides were incubatedwith primary polyclonal Ab in PBS-XG overnight at 4° C., washed 3 times5 min with PBS-XG, incubated with secondary conjugated polyclonal Ab orstreptavidin for 1 hour at room temperature, washed once, incubated with4′6-diamidino-2-phenylindole-2HCl (DAPI) (Sigma) 5 min at roomtemperature, washed 3 times 5 min and mounted with Fluoromount-G™(Southern Biotechnology associates). Slides were examined under anAxiolmager M1™ fluorescence microscope (Zeiss) equipped with a CCDcamera and images were processed with AxioVision™ software (Zeiss).

Antibodies.

Purchased from BD Biosciences: biotynilated anti-CD45.2.Purchased from Sigma: Cy3-conjugated anti-αSMA (1A4) and streptavidin.Purchased from Serotec: rabbit anti-mouse collagen 1A antibodies.Purchased from Invitrogen: purified rabbit anti-GFP (A-11122),FITC-conjugated anti-rabbit polyclonal antibodies, Alexa Fluor 488 or647-conjugated anti-rabbit IgG (H+L).Purchased from Andrew Farr (University of Washington, Seattle, Wash.Seattle): anti-gp38 culture supernatant.Purchased from Jackson Immunoresearch: Cy3 conjugated anti-syrianhamster.Purchased from Abcam: chicken anti-GFP polyclonal antibodies.

Chemicals.

CFA was purchased from Sigma. Cardiotoxin was purchased from Latoxan(Valence, France).

Results Generation of BAC Transgenic Mice ADAM12 CRE-IRES-EGFP

ADAM12 has been previously described to be overexpressed in a number ofpathologies, including muscle injury (Borneman et al., “Analysis fortranscript expression of meltrin alpha in normal, regenerating, anddenervated rat muscle”. J Muscle Res Cell Motil., 2000, 21:475-480,) andcancer (Peduto et al., “ADAM12 is highly expressed incarcinoma-associated stroma and is required for mouse prostate tumorprogression”. Oncogene. 2006 Aug. 31; 25(39):5462-6). To get a betterinsight into the development of ADAM12-expressing cells, the inventorsgenerated bacterial artificial chromosomes (BAC) transgenic miceexpressing Cre recombinase and the fluorescent reporter EGFP undercontrol of the Adam12 gene (Adam12-Cre-Ires-EGFPTG mice, referredthereafter as m12-CIG) (FIG. 1A). Given the large size of BACs, most ifnot all regulatory sequences are present in order to maximize chances todirect faithful and tissue-specific expression of the transgene. Toallow visualization of ADAM12 expression at the same time than cell fatemapping of these cells, M12-CIG mice were bred to R26R red fluorescentreporter mice, which express RFP under control of the ubiquitouslyactive gene Rosa26 after a LoxP-flanked STOP cassette is excised by Cre(Luche et al., “Faithful activation of an extra-bright red fluorescentprotein in “knock-in” Cre-reporter mice ideally suited for lineagetracing studies”. Eur J Immunol. 2007 January; 37(1):43-53). In thesemice (referred thereafter as M12cre/RFP mice, FIG. 1B), any cell thathad expressed ADAM12 will permanently be labelled with RFP.

ADAM12 Labels Stromal Cells Arising During CFA-Induced Skin Inflammation

ADAM12 is not expressed or expressed at very low levels in normal adulttissues, such as muscle, skin or nerve under homeostatic conditions. Todetermine whether ADAM12 may be involved in tissue injury andinflammation, the inventors induced skin inflammation by injectingComplete Freud Adjuvant (CFA) in the ear. The inventors observed thegeneration of an important population of ADAM12+ cells starting as soonas 24 h after injection of CFA. These cells expanded rapidly until 96 hand were predominantly found surrounding the muscle fibers at the baseof the ear (FIG. 2A, top panel; the green staining observed in the leftpanel—ear without CFA—is autofluorescence of the tissue and is used tobetter delineate the ear). To perform lineage tracing ofADAM12-expressing cells during inflammation, M12-CIG mice were crossedwith Rosa26+/FL RFP. In the resulting M12cre/RFP mice, the inventorsobserved that the large population of lymphoid gp38+ SMA+ stromaexpanding after CFA injection, which the inventors previouslycharacterized as being essential for leucocyte recruitment and survival(Peduto et al.,” Inflammation recapitulates the ontogeny of lymphoidstromal cells.” J Immunol. 2009 May 1; 182(9):5789-99), was RFP andtherefore was derived from precursor cells that had expressed ADAM12(FIG. 2A, lower panel and FIG. 2C, top panel). GFP+ expression decreasedrapidly after 4 days, however could still be detected in few RFP+ cells2 weeks after CFA-inflammation (FIG. 2A, lower panel).

Cell Fate Mapping of ADAM12-Expressing Cells During Muscle Inflammation

After injury, muscle healing occurs through different phases, includingmuscle degeneration and inflammation, muscle regeneration, anddevelopment of fibrosis. The inventors induced muscle injury andregeneration in m12-CIG mice by injecting cardiotoxin (CX) in theTibialis Anterior (TA) muscle. After a first phase of muscle damageinduced by the toxin and local inflammation, muscle stem cells becomeactivated to regenerate the muscle fibers in 2-3 weeks. A similar ADAM12expression than in skin inflammation was observed. GFP+ cells wererapidly detected in stromal cells filling the damaged and highlyinfiltrated regions of the muscle, with the strongest expression at day4-5 after cardiotoxin injection (FIG. 2B, top panel), which correspondsto the inflammatory phase (with high leucocytes infiltration). To allowvisualization of ADAM12 expression at the same time than cell fatemapping of their progeny, further experiments were performed in theM12cre/RFP mouse. After the first wave of high ADAM12 expression, GFP+cells decreased notably but were still detected in the stroma ofregenerating muscle 15 days after CX injection (FIG. 2B, lower panel).At the same time than the number of GFP+ cells decrease in the damagedregions, a massive increase of fate-mapped RFP+SMA+ cells was observed.After 3-4 weeks, the inventors observed that more than 50% of fibrosis(from 50% to 80%, as defined by myofibroblasts staining with alpha-SMAantibodies) was RFP, and therefore was progeny of precursors cells thathad expressed ADAM12 (FIG. 2B, lower panel; and FIG. 2C lower panel).

Altogether, these results show that ADAM12 is transiently overexpressedfollowing muscle and skin injuries and inflammation. The progeny ofADAM12+ cells (RFP+ cells) increase thereafter (FIG. 2D), making ADAM12a specific marker for fibrosis

ADAM12-Expressing Cells Express Pro-Inflammatory Genes

During inflammation, tissue-resident cells play a pivotal role byproviding inflammatory signals and growth factors. To get a betterunderstanding of the nature of ADAM12-expressing cells generated upontissue injury and inflammation, CD45-GFP+ and CD45-GFP-cells wereFACS-sorted at days 2-4 after cardiotoxin injection. Total RNA wasextracted from these subsets of stromal cells, and transcripts'expression was measured by using quantitative reverse transcriptase(qRT)-PCR.

The inventors observed that, compared to GFP-cells, GFP+ cellsup-regulated transcripts for IL1 beta, IL-6, and TNF-alpha, all withknown proinflammatory functions; TGF beta, a master gene involved inmyofibroblast differentiation; as well as CXCL1 and CXCL10, bothinvolved in leucocytes recruitment (FIG. 3A). In addition, theyexpressed high levels of pleiotropic growth factors CTGF (connectivetissue growth factor), IGF1 and FGF1, and of col1A responsible for thesynthesis of type I collagen deposited in the extracellular matrix (FIG.3B).

ADAM12 Knock Down In Vivo by RNA Interference DecreasesInflammation-Induced Fibrosis

To determine whether ADAM12 is directly involved in the generation offibrotic lesions occurring during inflammation, ADAM12 was knocked downin vivo in inflamed tissues by injecting ADAM12-specific siRNA(following a protocol described in Materials & Methods).

Ear skin or Tibialis Anterior muscles were first injected respectivelywith CFA and cardiotoxin to induce inflammation, and a solution ofsiRNA/invivofectamine specially formulated for in vivo delivery ofsiRNAs was injected locally at days 1 and 3 after CFA injection, and atdays 2/7/12 or 2/7/12/17 after CX injection. Control tissues wereinjected with scrambled siRNAs with similar GC content. The tissues werecollected at day 6 for CFA-ear and at day 22 for CX-muscle, andprocessed for histology or snapped-freezed for subsequent RNAextraction. The inventors observed that the accumulation of stromalextracellular matrix (as detected by staining with ERTR7 antibodies) wasdecreased locally in the tissues treated with ADAM12 siRNAs comparedwith tissues treated with scrambled siRNA (FIG. 4A). The lymphoidstromal cells expanding during inflammation were previouslycharacterized as expressing gp38 (Peduto et al., 2009). The inventorsalso observed a decrease in the population of gp38+ lymphoid stromalcells, and in accordance with a role for gp38+ cells in recruitingleucocytes, a decreased number of CD45+ cells were locally infiltratingthe tissues (FIG. 4A, a representative picture of three independentexperiments is shown). In agreement with the histology results,quantitative RT-PCR indicated a decrease in SMA expression (expressed bymyofibroblasts mainly responsible for fibrosis expansion); as well as adecrease in transcripts coding for type I collagen (col1A), CXCL1 andCXCL10; and CTGF and TGF beta, which represent essential players ofmyofibroblast development (FIG. 4B). The detected decrease in CXCL1 andCXCL10 in total tissue may be involved in the observed decrease inleucocyte recruitment. The decrease was more pronounced in the muscletissue after 22 days, even though it was already evident in the earafter 6 days. The qRTPCR results represent 3 independent experiments.

The inventors observed that the fibrotic index (as determined byaccumulation of collagen measured by staining with col1A antibodies) wasdecreased locally in muscles treated with ADAM12 siRNAs compared tomuscles treated with scrambled siRNA (FIG. 4C).

Ablation of ADAM12-Expressing Cells Decreases Inflammation and Fibrosis

M12tTA/LC1/YFP mice were generated that express tTA and the diphteriatoxin receptor (DTR) under control of the Adam12 locus on a BAC, as wellas the tTA-controlled expression of Cre (LC-1) and the reporterRosa26^(+/loxP-EYFP) locus (FIG. 6A). In these mice, the permanentYFP-labeling of the progeny of ADAM12⁺ cells is controlled in time bythe administration of doxycycline (dox), which blocks the tTA-mediatedlabeling cascade, allowing for a time-controlled fate mapping of ADAM12⁺cells. To formally demonstrate that cells overexpressing ADAM12 upontissue injury and their progeny are required for the fibrotic process,M12tTA/LC1/YFP mice were injected with diphtheria toxin subunit A (DTA)4, 7 and 10 days after cardiotoxin injection to induce the ablation ofADAM12⁺ cells. As expected, injection of DTA induced a marked reductionin the generation of YFP⁺ cells (FIG. 6B). Consistent with thepro-inflammatory nature of ADAM12⁺ cells (FIG. 3), leucocytesinfiltration was significantly decreased upon DTA treatment (FIG. 6B,CD45+ cells). As a consequence of the loss in ADAM12⁺ cells-derivedpro-fibrotic stroma, collagen accumulation was reduced in injuredmuscles (FIG. 6C).

Nerve Injury Triggers the Generation of ADAM12+ Cells

Damage to a nerve triggers degeneration of the axon distal to theinjury, as well as activation of stromal and inflammatory cells (Chen,Z. L., Yu, W. M. & Strickland, S. Peripheral regeneration. Annu RevNeurosci., 2007, 30, 209-233). Sciatic nerve injury alone induced, inthe innervated muscle, the transient development of GFP⁺ cells in thefirst days post-injury (FIG. 7A left panel), and of their cellularprogeny that co-localized with collagen deposits, 2-3 weeks post-injury(FIG. 7A right panel)

1. (canceled)
 2. The composition according to claim 5, wherein theinhibitor affects or inhibits ADAM12 gene expression or ADAM12 proteinactivity.
 3. The composition according to claim 5, wherein the inhibitoris an antisense oligonucleotide, a siRNA, a miRNA, a small organicmolecule, an enzyme, an antibody, a peptide or a polynucleotide encodinga polypeptide.
 4. The composition according to claim 5, wherein theinhibitor is a small interfering RNA specific for ADAM12.
 5. Acomposition for preventing or treating inflammation-induced fibrosiscomprising an ADAM12 inhibitor and a pharmaceutically acceptablecarrier.
 6. A method for preventing or treating inflammation-inducedfibrosis in a subject, comprising the step of administering thecomposition as defined in claim 5 to said subject.
 7. (canceled) 8.(canceled)
 9. A method for diagnosing inflammation-induced fibrosis in asubject, comprising: detecting the expression of ADAM12 gene in stromalcells from a biological sample of said subject, and relating theexpression of ADAM12 gene to the presence or the advent ofinflammation-induced fibrosis.
 10. A method for diagnosinginflammation-induced fibrosis in a subject, comprising: determining thelevel of ADAM12 gene expression in stromal cells from a biologicalsample of said subject, and comparing this level with a reference level,whereby an increased level of ADAM12 gene expression in said stromalcells with respect to the reference level indicating the presence or theadvent of inflammation-induced fibrosis.
 11. A method for detectingstromal cells activated upon tissue injury and inflammation and thatwill generate pro-fibrotic cells in a subject comprising: detecting theexpression of ADAM12 gene in the stromal cells; and relating theexpression of ADAM12 gene to the presence of these stromal cells. 12.The method according to claim 9, further comprising a step of isolatingstromal cells, before the step of detecting.
 13. An isolated bacterialstrain comprising a generated bacterial artificial chromosome (BAC)expressing a Cre recombinase and a fluorescent reporter EGFP undercontrol of the Adam12 gene, deposited on Aug. 28, 2009 under No I-4225at the CNCM.
 14. A transgenic mouse comprising the BAC contained in thebacterial strain according to claim
 13. 15. A screening method foridentifying ADAM12 inhibitors, comprising the steps of: inducing aninflammation response at a desired site of the transgenic mouse of claim14, applying at the site of inflammation a candidate compound to betested, evaluating expression of the fluorescent reporter EGFPpolypeptide; and identifying that the expression level of saidfluorescent reporter EGFP is inhibited, therefore indicating that thecandidate compound has the capacity of inhibiting ADAM12 expression. 16.A cytotoxic compound able to specifically kill ADAM12 expressing stromalcells for use to prevent or treat fibrosis, wherein said compoundcomprises a targeting molecule selected among an antibody or a smallinterfering RNA specific for ADAM12 according to claim 3, and acytotoxic molecule which is a toxin.
 17. A composition for preventing ortreating inflammation-induced fibrosis comprising the cytotoxic compoundof claim 16, and a pharmaceutically acceptable carrier.
 18. (canceled)19. The method according to claim 10, further comprising a step ofisolating stromal cells, before the step of detecting.
 20. The methodaccording to claim 11, further comprising a step of isolating stromalcells, before the step of detecting.